An analytical method allows to detect the possible presence of one or more analytes in a sample. An analytical method is generally performed on a solid support. Among the analytical methods, the multiplex analytical method allows to detect simultaneously the possible presence of several analytes within the same sample. A multiplex analytical method can be performed on a solid support comprising dots or else a plurality of beads.
Conventionally, an analytical method comprises a step of contacting a sample to be analyzed with at least one dot of a solid support or with beads comprising a specific capture ligand of an analyte to be detected, a step of adding a detection ligand specific for an analyte to be detected and coupled to a direct or indirect marker, any development step by addition of a reporter itself coupled to a direct or indirect marker, and a signal detection step (also called signal acquisition step). In the case of an indirect marker of the peroxidase enzyme type, adding a substrate of the enzyme allows an enzymatic reaction which leads to the production of a chemiluminescent compound. The signal is then detected by chemiluminescence.
Detecting a chemiluminescent signal requires, in principle, to perform signal acquisition in the presence of the substrate of the enzyme, i.e., in the presence of a liquid phase, in order to allow the continuous production of chemiluminescent compound.
Indeed, if a washing step is performed before the signal acquisition, the residual substrate is removed and the enzymatic reaction is stopped. However, it is known that the signal emitted by the chemiluminescent compound gradually fades away. Accordingly, the substrate of the enzyme must be present in a liquid phase in contact with the solid phase to allow a sufficiently stable and reproducible emission of the signal.
However, the acquisition of a signal at the dots of a well of a microplate in the presence of a liquid phase leads to light interference. Such light interference has several origins: first, photons emitted from dots close to the top of the well can interact with the compounds of the solution comprising the chemiluminescent compound and be disseminated in all directions; on the other hand, the photons may also be reflected by the walls of the well and the change of medium at the liquid/air interface, more precisely at the level of the meniscus formed by the interaction of the well wall and the solution comprising the chemiluminescent compound.
Such light interference can produce dots called “twin dots”, slightly offset from the actual dots, a visible light ring on the periphery of the well, or even a light arc when the signal emitted at a dot is strong. Such light interference thus induces a problematic background noise, which can be the source of false negative or false positive results. For example, the light ring around the well can bias the background noise threshold, a weak signal then being drowned in the background noise. Light interference can also interfere with the verification of the absence of a failure in a dot effected by an annular measurement around a dot.
The document EP 0 165 072 discloses the use of an attenuator, preferably a mixture of red and yellow dyes, to suppress unwanted light and false positives results in a process involving a luminescent light emission. In this document, the unwanted light is adjacent to the measured surfaces. The test is performed in a hollow elongated pipette comprising a plurality of cotton yarns, each cotton yarn being linked to one type of antigen. The pipette is closed with a transparent window. The signal is revealed in the dark by pressing a film onto the surface of the pipette. The attenuator allows the suppression of false positives associated with non-specific binding of the HRP peroxidase enzyme to the cotton yarns.
Document U.S. Pat. No. 8,163,562 describes a test allowing to reduce undesirable light resulting from fluorescence of a solution in which is immersed a cellular compartment, which is preferably a cell. This undesired fluorescence originates especially from probes or chemical compounds used during the test. The signal to be detected is derived in turn from a photon producing agent located in the membrane compartment. For this purpose, a photon reducing agent impermeable to the membrane and not specifically binding to the membrane is used in the aqueous solution in contact with the outer surface of the membrane compartment. The photon reducing agent may be a dye or mixture of dyes.
There is therefore a need for solutions allowing to improve the detection of a signal corresponding to the presence of an analyte in the context of an analytical method performed on a dot or dots in which the acquisition of the signal at the dot or dots occurs in the presence of a liquid phase in order to secure the results obtained, avoiding false positive or false negative results.